During the last grant period, we discovered that chronic ethanol feeding causes the development and maintenance of fatty liver in mice by affecting several important liver transcription factors and co-factors; namely, sterol regulatory element binding protein 1 (SREBP-1), peroxisome proliferator-activated receptor alpha (PPAR-alpha) and PPAR-gamma co-activator-alpha (PGC-1-alpha.) We demonstrated that the effects of ethanol on these transcriptional regulators are mediated partially by inhibition of hepatic AMP-activated kinase (AMPK), a key metabolic switch that controls pathways of hepatic lipid metabolism. It is intriguing that ethanol metabolism is required for each of these effects to occur, as ethanol metabolism has also been unequivocally associated with the development of alcoholic fatty liver disease. However, the identity of a potential bridging molecule that might link ethanol metabolism with downstream effects (including activation of said transcriptional regulators and the expression of genes that ultimately promote lipid accumulation) is still unknown. Sirtuin 1 (SIRT1), a class III NAD+-dependent protein deacetylase, is emerging as a master lipid regulator. The requirement of NAD+ for SIRT1 enzymatic activity implies a potential link between ethanol metabolism and SIRT1. Therefore, the objective of the current proposal is to test the hypothesis that ethanol metabolism significantly down regulates SIRT1 in the liver. Such inhibition of SIRT1 may lead to impairment of hepatic fat metabolism through modulation of the above-described signaling molecules, thereby contributing to increased hepatic lipid synthesis and reduced oxidation and export of fatty acids. This hypothesis will be tested in both animal and cell culture models of chronic ethanol exposure. In the mouse liver, we will examine the effects of chronic ethanol feeding on SIRT1--in terms of its mRNA and protein levels, enzymatic activity, and responsiveness to a potent SIRT1 agonist (resveratrol). The effects of ethanol will be correlated with hepatic lipid content and histology. In primary cultured hepatocytes and in hepatoma cells, the molecular mechanisms by which ethanol metabolism inhibits SIRT1 and alters signaling events will be investigated. Since SIRT1 can be manipulated by both pharmacological agents and dietary polyphenols, testing this hypothesis could possibly lead to novel therapies for human alcoholic fatty liver disease and steatohepatitis.